Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is a major cause of morbidity and mortality in immune compromised individuals. HCMV undergoes a lytic infection, which is characterized by transcriptional activation that involves the entire genome leading to the production of infectious virus. HCMV, like all herpesviruses, establishes a life-long latency with its host. Viral latency is marked by the production of a limited number of transcripts in the absence of immediate early gene expression and the persistence of the viral genome in latently infected cells. HCMV latency was shown to occur in myeloid cells (CD34+) and monocytes (CD14+). To date, almost nothing is known about the mechanisms controlling HCMV latency. Although a few latency-associated transcripts were identified, the relationship of these factors to maintenance and/or replication of the viral chromosome in latently infected cells are not established. The long-term goal for this proposal is to identify viral encoded factors that directly contribute to maintenance or replication of the virl genome such that strategies can be developed to purge the latent virus from cells. The objective for this application is to define the cis acting virus sequence(s) that mediate maintenance/replication of the virus genome, and elucidate the trans acting factors that regulate viral genome persistence. The central hypothesis is that viral cis acting sequences and trans acting factors mediate the persistence of the viral chromosome during latency. This hypothesis was formulated based on preliminary data generated in the applicants' laboratory. The rationale for the proposed research is that the understanding of the mechanism(s) involved in maintenance of the viral genome has the potential to lead to better therapies for the treatment of HCMV-related disease. Guided by strong preliminary data, this hypothesis will be tested by pursuing 3 specific aims: 1) Elucidate the role of IE1 and other viral and cellular factors in maintenance of the HCMV viral genome in latently infected CD34 (+) cells. Our recently published data identified latency associated transcripts in experimentally and naturally latently infected CD14 (+) and CD34 (+) cells. Some of these transcripts encode factors previously shown to be involved in DNA replication and viral DNA stability. We also show that a plasmid containing the terminal (TR) repeat region of the virus genome persists in latently infected CD14 (+) monocytes as well as CD34 (+) hematopoietic cells. 2) Develop transient replication assays to define specific viral DNA motifs required for maintenance of the TR element. The subcloned TR element will be used to develop a transient assay to evaluate essential cis acting sequences. The third aim will explore the role of trans acting factors in viral genome maintenance using the TR plasmid as a reporter for maintenance/replication. The approach is innovative because it utilizes RNA-Seq and nucleosome depletion data to identify essential cis and trans acting factors that contribute to HCMV latency. The proposed research is significant because it is expected that we will elucidate the mechanism involved in HCMV latency and potentially lead to novel approaches to treat infection.